EP4279509A1 - Garp protein antibody and application thereof - Google Patents

Garp protein antibody and application thereof Download PDF

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Publication number
EP4279509A1
EP4279509A1 EP22739160.4A EP22739160A EP4279509A1 EP 4279509 A1 EP4279509 A1 EP 4279509A1 EP 22739160 A EP22739160 A EP 22739160A EP 4279509 A1 EP4279509 A1 EP 4279509A1
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Prior art keywords
seq
amino acid
acid sequence
set forth
binding protein
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP22739160.4A
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German (de)
English (en)
French (fr)
Inventor
Jianjian ZHANG
Zhongzong PAN
Xinxiu YANG
Xiaowu Liu
Lu Yang
Peipei LIU
Xiaodan CAO
Sujun DENG
Xueping Wang
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Jiangxi Jemincare Group Co Ltd
Shanghai Jemincare Pharmaceuticals Co Ltd
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Jiangxi Jemincare Group Co Ltd
Shanghai Jemincare Pharmaceuticals Co Ltd
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Publication of EP4279509A1 publication Critical patent/EP4279509A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
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    • A61K39/46—Cellular immunotherapy
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
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    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to the field of biomedicine, and in particular to a GARP protein antibody and use thereof.
  • Tregs are closely associated with autoimmune diseases and tumorigenesis, the abnormal activity of which can cause autoimmune diseases or tumorigenesis.
  • Treg cells as a current research hotspot in the field of immunology have two characteristics of low immune response and immunosuppression, and play a role in negative immunoregulation by inhibiting an immune system in an "active" mode.
  • Tregs can be classified into naturally occurring CD4+CD25+Tregs (natural regulatory T cells, nTregs) and induced adaptive regulatory T cells (aTregs or iTregs).
  • TGF- ⁇ forms a cage structure with the inactive binding protein LAP in an inactive state, which can be activated by GARP, integrins, proteases or other substrates to form active TGF- ⁇ , which activates cellular signaling by binding to receptors via autocrine or paracrine.
  • TGF- ⁇ signaling is thought to be primarily involved in the development of tumors resistance to checkpoint blockade therapies. For example, immunosuppressive cells and immunosuppressive factors are increased by TGF- ⁇ in the tumor microenvironment.
  • TGF- ⁇ can inhibit the immune response broadly through different mechanisms.
  • TGF- ⁇ reduces the differentiation and function of Th1, Th2 cells, and cytotoxic T lymphocytes (CTLs), all of which provide important anti-tumor effects.
  • TGF- ⁇ also enhances immune tolerance and tumor evasion by regulating the number and function of regulatory T (Treg) cells.
  • TGF- ⁇ regulates immune cell fate by inhibiting or stimulating cell proliferation, thereby affecting the development of the thymus and peripheral T cells.
  • the present application provides an isolated antigen-binding protein having one or more of the following properties: 1) binding to the human GARP/human TGF- ⁇ 1 complex in an Octet assay with a KD value of about 1.0E-12 or less; 2) not binding to cells expressing only human GARP or cells expressing only human TGF- ⁇ 1; 3) inhibiting the SMAD2 phosphorylation in Treg cells; 4) inhibiting the release of TGF- ⁇ 1 from HEK293T cells expressing human GARP/human TGF- ⁇ 1; 5) preventing Treg cells from inhibiting the release of interleukin-2 (IL-2) and interferon- ⁇ (IFN- ⁇ ) from human peripheral blood mononuclear cells (PBMC); and 6) blocking the inhibitory effect of Treg cells on GvHD induced by the human PBMCs in a human PBMC transplanted mice GvHD model.
  • the present application provides an isolated antigen-binding protein comprising HCDR3 comprising an amino acid sequence as set forth in
  • the isolated antigen-binding protein comprises HCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 3.
  • the isolated antigen-binding protein comprises HCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 2.
  • the isolated antigen-binding protein comprises a heavy chain variable region VH comprising the HCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 4.
  • the isolated antigen-binding protein comprises H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises an amino acid sequence as set forth in SEQ ID NO: 46.
  • the H-FR1 of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 5 and SEQ ID NO: 22.
  • the isolated antigen-binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises an amino acid sequence as set forth in SEQ ID NO: 47.
  • the H-FR2 of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 6 and SEQ ID NO: 23.
  • the isolated antigen-binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises an amino acid sequence as set forth in SEQ ID NO: 48.
  • the H-FR3 of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 7 and SEQ ID NO: 24.
  • the isolated antigen-binding protein comprises H-FR4, wherein the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises an amino acid sequence as set forth in SEQ ID NO: 49.
  • the H-FR4 of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 8 and SEQ ID NO: 25.
  • the isolated antigen-binding protein comprises H-FR1 comprising an amino acid sequence as set forth in SEQ ID NO: 46, H-FR2 comprising an amino acid sequence as set forth in SEQ ID NO: 47, H-FR3 comprising an amino acid sequence as set forth in SEQ ID NO: 48, and H-FR4 comprising an amino acid sequence as set forth in SEQ ID NO: 49.
  • the isolated antigen-binding protein comprises H-FR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 5 and SEQ ID NO: 22, H-FR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6 and SEQ ID NO: 23, H-FR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 7 and SEQ ID NO: 24, and H-FR4 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 8 and SEQ ID NO: 25.
  • the H-FR1, H-FR2, H-FR3, and H-FR4 of the antigen-binding protein comprise amino acid sequences selected from any one of the groups consisting of:
  • the isolated antigen-binding protein comprises a heavy chain variable region VH comprising an amino acid sequence as set forth in SEQ ID NO: 50.
  • the VH of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 1 and SEQ ID NO: 21.
  • the isolated antigen-binding protein comprises LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein comprises LCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 45.
  • the isolated antigen-binding protein comprises LCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 and SEQ ID NO: 28.
  • the isolated antigen-binding protein comprises LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 44.
  • the isolated antigen-binding protein comprises LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 and SEQ ID NO: 27.
  • the isolated antigen-binding protein comprises a light chain variable region VL which comprises the LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 44, the LCDR2 having amino acid sequence as set forth in SEQ ID NO: 45, and the LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein comprises a light chain variable region VL comprising the LCDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 10 and SEQ ID NO: 27, the LCDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 11 and SEQ ID NO: 28, and the LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12.
  • the LCDR1, LCDR2, and LCDR3 in the isolated antigen-binding protein have an amino acid sequence selected from any one of the groups consisting of:
  • the isolated antigen-binding protein comprises L-FR1, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises an amino acid sequence as set forth in SEQ ID NO: 52.
  • the L-FR1 of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 13 and SEQ ID NO: 29.
  • the isolated antigen-binding protein comprises L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises an amino acid sequence as set forth in SEQ ID NO: 14.
  • the isolated antigen-binding protein comprises L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises an amino acid sequence as set forth in SEQ ID NO: 53.
  • the L-FR3 of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 15 and SEQ ID NO: 30.
  • the isolated antigen-binding protein comprises L-FR4, wherein the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises an amino acid sequence as set forth in SEQ ID NO: 54.
  • the L-FR4 of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 16 and SEQ ID NO: 31.
  • the isolated antigen-binding protein comprises L-FR1 comprising an amino acid sequence as set forth in SEQ ID NO: 52, L-FR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14, L-FR3 comprising an amino acid sequence as set forth in SEQ ID NO: 53, and L-FR4 comprising an amino acid sequence as set forth in SEQ ID NO: 54.
  • the isolated antigen-binding protein comprises L-FR1 comprising an amino acid as sequence as set forth in any one of SEQ ID NO: 13 and SEQ ID NO: 29, L-FR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14, L-FR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 15 and SEQ ID NO: 30, and L-FR4 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 16 and SEQ ID NO: 31.
  • the L-FR1, L-FR2, H-FR3, and L-FR4 of the antigen-binding protein comprise amino acid sequences selected from any one of the groups consisting of:
  • the isolated antigen-binding protein comprises a light chain variable region VL comprising an amino acid sequence as set forth in SEQ ID NO: 51.
  • the VL of the antigen-binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 9 and SEQ ID NO: 26.
  • the isolated antigen-binding protein comprises a heavy chain constant region comprising an IgG-derived constant region or an IgY-derived constant region.
  • the antigen-binding protein heavy chain constant region comprises a human IgG4-derived constant region.
  • the antigen-binding protein heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 17.
  • the isolated antigen-binding protein comprises a light chain constant region, and wherein the antibody light chain constant region comprises an Ig ⁇ -derived constant region or an Ig ⁇ -derived constant region.
  • the antigen-binding protein light chain constant region comprises a human Ig ⁇ -derived constant region.
  • the antigen-binding protein light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18.
  • the isolated antigen-binding protein comprises a heavy chain HC comprising an amino acid sequence as set forth in any one of SEQ ID NO: 19 and SEQ ID NO: 32.
  • the isolated antigen-binding protein comprises a light chain LC comprising an amino acid sequence as set forth in any one of SEQ ID NO: 20 and SEQ ID NO: 33.
  • the isolated antigen-binding protein comprises an HC comprising an amino acid sequence as set forth in any one of SEQ ID NO: 19 and SEQ ID NO: 32 and an LC comprising an amino acid sequence as set forth in any one of SEQ ID NO: 20 and SEQ ID NO: 33.
  • the HC and LC of the antigen-binding protein comprise amino acid sequences selected from any one of the groups consisting of:
  • the isolated antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the antibody is selected from the group consisting of: monoclonal antibodies, single chain antibodies, murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the present application provides a polypeptide comprising the isolated antigen-binding protein.
  • the present application provides an immunoconjugate comprising the isolated antigen-binding protein or the polypeptide.
  • the immunoconjugate also includes a pharmaceutically acceptable therapeutic agent.
  • the therapeutic agent is selected from the group of a cytotoxic agent and a cytostatic agent.
  • the present application provides an isolated nucleic acid molecule encoding the isolated antigen-binding protein, or the polypeptide.
  • the present application provides a vector comprising the isolated nucleic acid molecule.
  • the present application provides a cell comprising the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, and/or the vector.
  • the present application provides a method of preparing the isolated antigen-binding protein or the polypeptide, the method comprising culturing the cell under conditions capable of expressing the isolated antigen-binding protein or the polypeptide.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable adjuvant and/or excipient.
  • the present application provides a pharmaceutical combination comprising the isolated antigen-binding protein and an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor comprises a substance that inhibits the interaction of PD-1/PD-L1.
  • the immune checkpoint inhibitor is selected from the group consisting of: PD-1/PD-L1 blocking agents, PD-1 antagonists, PD-L1 antagonists, PD-1 inhibitors, and PD-L1 inhibitors.
  • the immune checkpoint inhibitor comprises an anti-PD-L1 antibody.
  • the anti-PD-L1 antibody comprises HCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 37.
  • the anti-PD-L1 antibody comprises HCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 36.
  • the anti-PD-L1 antibody comprises HCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 35.
  • the anti-PD-L1 antibody comprises a heavy chain variable region VH comprising HCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 35, HCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 36, and HCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 37.
  • the anti-PD-L1 antibody comprises a heavy chain variable region VH comprising an amino acid sequence as set forth in SEQ ID NO: 34.
  • the anti-PD-L1 antibody comprises LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 41.
  • the anti-PD-L1 antibody comprises LCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 40.
  • the anti-PD-L1 antibody comprises LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 39.
  • the anti-PD-L1 antibody comprises a light chain variable region VL comprising LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 39, LCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 40, and LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 41.
  • the anti-PD-L1 antibody comprises a light chain variable region VL comprising an amino acid sequence as set forth in SEQ ID NO: 38.
  • the anti-PD-L1 antibody includes Atezolizumab.
  • the pharmaceutical combination can be a pharmaceutical composition.
  • the present application provides a kit comprising the pharmaceutical combination.
  • the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition for the prevention, remission, and/or treatment of a tumor.
  • the tumor includes a solid tumor.
  • the tumor includes a tumor associated with the expression of GARP.
  • the tumor includes a melanoma, a breast tumor, and/or a lung tumor.
  • the present application provides the pharmaceutical combination and/or the kit for the prevention, remission, and/or treatment of a tumor.
  • the tumor includes a solid tumor.
  • the tumor includes a tumor associated with the expression of GARP.
  • the tumor includes a melanoma, a breast tumor, and/or a lung tumor.
  • the present application provides a use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition in the manufacture of a medicament for the prevention, remission, and/or treatment of a tumor.
  • the tumor includes a solid tumor, a breast tumor, and/or a lung tumor. In some embodiments, the tumor includes a tumor associated with the expression of GARP.
  • the tumor includes a melanoma.
  • the present application provides a use of the pharmaceutical combination and/or the kit in the manufacture of a medicament for the prevention, remission, and/or treatment of a tumor.
  • the tumor includes a solid tumor.
  • the tumor includes a tumor associated with the expression of GARP.
  • the tumor includes a melanoma, a breast tumor, and/or a lung tumor.
  • the present application provides a method for the prevention and/or treatment of a disease or disorder comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the isolated nucleic acid molecule, the vector, the cell, and the pharmaceutical composition, wherein the disease or disorder includes a tumor.
  • the tumor includes a solid tumor.
  • the tumor includes a tumor associated with the expression of GARP.
  • the tumor includes a melanoma, a breast tumor, and/or a lung tumor.
  • the present application provides a method for the prevention and/or treatment of a disease or disorder comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination, wherein the disease or disorder includes a tumor.
  • the tumor includes a solid tumor.
  • the tumor includes a tumor associated with the expression of GARP.
  • the tumor includes a melanoma, a breast tumor, and/or a lung tumor.
  • isolated generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or the substance may be isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally occurs in a living animal and the same polynucleotide or polypeptide isolated from its natural state in high purity is the to be isolated.
  • isolated does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • the term “antigen-binding protein” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen.
  • the term “antigen-binding protein” may include an "antibody” or an "antigen-binding fragment".
  • the antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and may include any molecule comprising an antigen-binding portion thereof.
  • antibody may include monoclonal antibodies, antibody fragments, or antibody derivatives, including, but not limited to, murine antibodies, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., Fab, Fab', and (Fab)2 fragments).
  • antibody may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain may be composed of a heavy chain variable region and a heavy chain constant region.
  • Each light chain may be composed of a light chain variable region and a light chain constant region.
  • the VH and VL regions can be further distinguished as hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL may be composed of three CDRs and four FRs, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with an antigen (e.g., human GARP).
  • the constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).
  • CDRs The exact boundaries of the CDRs have been defined differently for different systems.
  • the system described by Kabat Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 ) and (1991)) provides not only an unambiguous residue numbering system applicable to any variable region of an antigen-binding fragment, but also provides precise residue boundaries defining CDRs. These CDRs may be referred to as Kabat CDRs.
  • Chothia and co-workers found that certain sub-portions within Kabat CDRs adopt nearly identical conformations in the backbone of a peptide despite the large diversity at the amino acid sequence level. These sub-portions are designated as L1, L2, and L3 or H1, H2, and H3, where "L” and "H” refer to the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
  • the term "antigen-binding fragment” generally refers to one or more fragments of an antibody that function to specifically bind to an antigen.
  • the antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody.
  • the antigen-binding function of an antibody may also be achieved by: a heavy chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab' or F (ab')2, or a light chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab', or F(ab')2.
  • An Fab fragment i.e., a monovalent fragment consisting of the VL, VH, CL, and CH domains
  • an F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region
  • an Fd fragment consisting of the VH and CH domains
  • an Fv fragment consisting of the VL and VH domains of a single arm of an antibody
  • a dAb fragment consisting of VH domains ( Ward et al., (1989) Nature 341: 544-546 )
  • an isolated complementarity determining region (CDR) and (7) a combination of two or more isolated CDRs which may optionally be joined by a linker.
  • scFv monovalent single chain molecules Fv formed by the pairing of VL and VH
  • scFv monovalent single chain molecules
  • a class of antibody VHH lacking the antibody light chain but only the heavy chain variable region can also be included (see, e.g., Kang Xiaozhen et al., Chinese Journal of Biotechnology, 2018, 34 (12): 1974-1984 ).
  • the "antigen-binding portion” may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • the term "monoclonal antibody” generally refers to a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • the monoclonal antibodies can be prepared by hybridoma techniques or can be produced in bacteria, eukaryotic animal or plant cells using recombinant DNA methods, or can be derived from phage antibody libraries using the techniques, for example, described in Clackson et al., Nature, 352:624-628 (1991 ) and Marks et al., Mol.Biol., 222:581-597 (1991 ).
  • chimeric antibody generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to the corresponding sequence in another species.
  • variable regions of both the light and heavy chains are derived from the variable region of an antibody of one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human).
  • non-human-derived B cells or hybridoma cells can be used to generate variable regions, while which the constant regions in combination therewith are of human origin.
  • variable region has the advantage of being easy to prepare and its specificity is not influenced by the origin of the constant region with which it is combined. Also, since the constant region of the chimeric antibody can be derived from human, the chimeric antibodies are less likely to elicit an immune response upon injection than using antibodies whose constant regions are of non-human origin.
  • humanized antibody generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of a xenogenous antibody when introduced into humans, while maintaining the full antigen-binding affinity and specificity of the antibody.
  • non-human binding domains can be humanized using the technical means, such as CDR transplantion ( Jones et al., Nature 321:522(1986 )) and variants thereof; including “reshaping", ( Verhoeyen, et al., 1988 Science 239:1534-1536 ; Riechmann, et al., 1988 Nature 332:323-337 ; Tempest, et al., Bio/Technol 1991 9:266-271 ), "hyperchimerization” ( Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033 ; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873 ; Co, et al., 1992 J Immunol 148:1149-1154 ), and “veneering” ( Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.”
  • CDR transplantion Jones
  • murine antibody generally refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises constant regions, which are also derived from mouse germline immunoglobulin sequences.
  • the murine antibodies of the present application may comprise amino acid residues not encoded by the mouse germline immunoglobulin sequences, such as mutations introduced by random or point mutations in vitro or by somatic mutation in vivo. However, the term “murine antibody” does not comprise the antibodies having CDR sequences from other mammalian species inserted into the framework sequences of a mouse.
  • GARP protein or “GARP antigen” are used interchangeably and include any variants and homologues of GARP.
  • the GARP is expressed naturally by a cell or is expressed by a cell transfected with a GARP gene.
  • GARP can be a human GARP having an accession number Q14392 in UniProt/Swiss-Prot.
  • GARP can be expressed on the surface of immune cells. For example, it can be expressed on the surface of regulatory T cells (Treg).
  • TGF- ⁇ 1 and “TGF ⁇ 1” are used interchangeably and generally associated with cell growth regulation and differentiation.
  • the TGF- ⁇ 1 is an isoform of TGF- ⁇ .
  • the "TGF- ⁇ 1” may include any variants and homologues of TGF- ⁇ 1 that are expressed naturally by a cell or in a cell transfected with a TGF- ⁇ 1 gene.
  • the "TGF- ⁇ 1” may be human TGF- ⁇ 1, which has accession number P01137 in UniProt/Swiss-Prot.
  • the "TGF- ⁇ 1" can be activated by GARP and binds to the TGF ⁇ receptor on CD8+T cells to inhibit the killing function of CD8+T cells on tumor cells.
  • the present application may also include functional variants, derivatives, analogs, homologs, and fragments thereof.
  • a variant of any given sequence refers to a sequence in which the particular sequence of residues, whether amino acid or nucleotide residues, has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function.
  • Variant sequences may be obtained by the addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the original functional activity is retained.
  • derivative generally refers to a polypeptide or polynucleotide of the present application comprising any substitution, variation, modification, replacement, deletion and/or addition of one (or more) amino acid residues from/on the sequence, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analog generally refers to a polypeptide or polynucleotide including any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.
  • amino acid substitutions e.g., at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitution can be made, so long as the modified sequence substantially retains the desired activity or ability.
  • Amino acid substitutions can include the use of non-naturally occurring analogs.
  • homologue generally refers to an amino acid sequence or nucleotide sequence having certain homology to a naturally occurring sequence.
  • the term “homology” can be equivalent to sequence "identity”.
  • homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence.
  • the homologue will comprise the same active site or the like as the subject amino acid sequence.
  • Homology may be considered in terms of similarity (i.e., amino acid residues having similar chemical properties/functions) or may be expressed in terms of sequence identity.
  • sequence having percent identity in any one of the SEQ ID NO of a mentioned amino acid sequence or nucleotide sequence refers to a sequence having the percent identity over the entire length of the mentioned SEQ ID NO.
  • sequence alignments may be performed by a variety of ways known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software, etc. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithm required to achieve the optimal alignment over the full length of the sequence being compared.
  • proteins or polypeptides used in the present application may also have deletions, insertions, or substitutions of amino acid residues which produce silent changes and result in functionally equivalent proteins.
  • Intentional amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids containing uncharged polar head groups with similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.
  • the term "tumor” generally refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors.
  • the tumor may include a solid tumor.
  • the tumor may include a tumor associated with protein expression of GARP.
  • the term "tumor associated with protein expression of GARP” generally refers to a tumor in which GARP expression leads to disease progression or evades immune surveillance.
  • the tumor associated with protein expression of GARP can be a GARP positive tumor.
  • the protein expression amount of GAPR on the surface of the tumor cells is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than that of normal cells.
  • the tumor may be a metastatic colon cancer.
  • the tumor may be a hepatocellular carcinoma.
  • the tumor may be an advanced renal cell carcinoma.
  • the tumor may be a non-small cell lung cancer.
  • the tumor may be a melanoma, a breast tumor, and/or a lung tumor.
  • melanoma generally refers to a highly malignant tumor of melanocytes origin. In the present application, the melanoma can occur in the skin as well as in the mucosa and viscera.
  • the term "immunoconjugate” generally refers to a conjugate formed by the conjugation of other agents (e.g., a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent) to the isolated antigen-binding protein (e.g., via covalent attachment of a linking molecule), wherein the conjugate can deliver the other agents to a target cell (e.g., a tumor cell) via specific binding of the isolated antigen-binding protein to an antigen on the target cell.
  • agents e.g., a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent
  • the immunoconjugate then undergoes such internalization and eventually enters the interior of the target cell (e.g., into vesicles such as a lysosome), at which point the linker molecule in the immunoconjugate can be cleaved to release the other agent, thereby exerting its cytotoxic effect.
  • the antigen may also be secreted by the target cell and located in the space outside the target cell.
  • the term "pharmaceutically acceptable therapeutic agent” generally refers to an agent that can inhibit the proliferation of tumors and/or tumor cells.
  • the therapeutic agent may be a cytotoxic agent or a cytostatic agent.
  • the therapeutic agent may be selected from the group consisting of: mitotic inhibitors, kinase inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylase inhibitors, anti-survival agents, and biological response modifiers.
  • the term "subject” generally refers to human or non-human animals, including, but not limited to, cats, dogs, horses, pigs, cows, caprid, rabbits, mice, rats, or monkeys.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides, or ribonucleotides or analogs thereof, of any length, isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host.
  • the vector can transfer the inserted nucleic acid molecule into and/or between cells.
  • the vector may include a vector for primarily inserting DNA or RNA into a cell, a vector for primarily replicating DNA or RNA, and a vector for primarily expressing transcription and/or translation of DNA or RNA.
  • the vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into an appropriate cell.
  • the vector may produce the desired expression product by culturing an appropriate cell containing the vector.
  • the vector may include a lentiviral vector.
  • the term "cell” generally refers to an individual cell, cell line or cell culture that may or may already contain a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing a polypeptide as described herein or an antigen-binding protein as described herein.
  • the cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutations, the progeny cells may not necessarily be identical in morphology or in genome to the original parent cell, but are capable of expressing the polypeptide or antigen-binding protein as described herein.
  • the cells can be obtained by transfecting cells in vitro with the vectors as described herein.
  • the cells may be prokaryotic cells (e.g., E.
  • the cells may be immune cells.
  • the immune cell may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells.
  • the immune cells may be T cells.
  • treatment generally refers to: (i) the prevention of the development of a disease, disorder, and/or condition in a patient who may be susceptible to, but has not yet been diagnosed with, that disease, disorder, or condition; (ii) the suppression of the disease, disorder, or condition, i.e., the curb of the development; and (iii) remission of the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition and/or symptoms associated with the disease, disorder, and/or condition.
  • polypeptide In the present application, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably and generally refer to a polymer of amino acids of any length.
  • the polymer may be linear or branched, and may comprise modified amino acids and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may comprise: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (e.g., binding to a labeling component).
  • amino acid includes natural and/or non-natural or synthetic amino acids, including glycine as well as the D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide refers generally to polymeric forms of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may have any three-dimensional structure and may perform any function that is known or unknown.
  • Non-limiting examples of polynucleotides are as follows: a coding or noncoding region of a gene or gene fragment, a plurality of loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transport RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • transport RNA transport RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozymes cDNA
  • recombinant polynucleotides branched polynucleo
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure may be performed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotides may be further modified after polymerization, e.g., by conjugation to labeled components.
  • K D (likewise, “K D “ or “KD ”) generally refers to an "affinity constant” or an “equilibrium dissociation constant” and refers to a value obtained at equilibrium in a titration measurement, or by dividing the dissociation rate constant (k d ) by the binding rate constant (k a ).
  • the binding affinity of a binding protein e.g., an isolated antigen-binding protein as described herein
  • an antigen e.g., a GARP protein
  • K D equilibrium dissociation constant
  • the K D value can be determined by Octet assay, and other experimental approaches and instruments such as BIAcore (Biomolecular Interaction Analysis) can be used (e.g., instruments available from BIAcoreInternationalAB, aGEHealthcarecompany, Uppsala, Sweden).
  • BIAcore Biomolecular Interaction Analysis
  • the K D value can be determined using KinExA (dynamic exclusion assay (KineticExclusionAssay)) available from SapidyneInstruments (Boise, Idaho) or using a surface plasmon resonance (SPR) instrument.
  • the term "about” generally refers to a range from 0.5% to 10% above or below the specified value, for example, a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.
  • the term "including” generally means comprising, summing up, encompassing or covering. In certain instances, the meaning of "being”, “consisting of" is also indicated.
  • the present application provides an isolated antigen-binding protein that can bind to a human GARP/human TGF- ⁇ 1 complex with a K D value of about 1.0E-12M or less (e.g., the K D is no greater than about 1.0E-12M, no greater than about 0.9E-12M, no greater than about 0.8E-12M, no greater than about 0.7E-12M, no greater than about 0.6E-12M, no greater than 0.5E-12M, no greater than 0.4E-12M, no greater than 0.3E-12M, no greater than 0.2E-12M, or no greater than 0.1E-12M or less).
  • the K D is no greater than about 1.0E-12M, no greater than about 0.9E-12M, no greater than about 0.8E-12M, no greater than about 0.7E-12M, no greater than about 0.6E-12M, no greater than 0.5E-12M, no greater than 0.4E-12M, no greater than 0.3E-12M, no greater than 0.2E-12M, or no greater than 0.1E-12M
  • the isolated antigen-binding protein may compete for binding to the human GARP/human TGF- ⁇ 1 complex with a reference antibody, which may comprise a heavy chain variable region VH, which may comprise at least one, two, or three of HCDR1, HCDR2, and HCDR3.
  • a reference antibody which may comprise a heavy chain variable region VH, which may comprise at least one, two, or three of HCDR1, HCDR2, and HCDR3.
  • the HCDR3 of the reference antibody may include an amino acid sequence as set forth in SEQ ID NO: 4.
  • the HCDR2 of the reference antibody may include an amino acid sequence as set forth in SEQ ID NO: 3
  • the HCDR1 of the reference antibody may include an amino acid sequence as set forth in SEQ ID NO: 2.
  • the reference antibody may include Antibody 8H2D7B3 or an antibody having the same HCDR1-3 as Antibody 8H2D7B3.
  • the reference antibody may include Antibody JYB1907hz0 or an antibody having the same HCDR1-3 as Antibody JYB 1907hz0.
  • the reference antibody may include Antibody JYB1907hz18 or an antibody having the same HCDR1-3 as Antibody JYB1907hz18.
  • the VH of the reference antibody may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the H-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 46.
  • the H-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 22.
  • the H-FR2 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 47.
  • WMYWVX 1 QX 2 PX 3 QGLEWIGSI (SEQ ID NO: 47), wherein X 1 may be K or R, X 2 may be A or R, and X 3 may be G or I.
  • the H-FR2 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 23.
  • the H-FR3 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 48.
  • THYNQKFX 1 X 2 RX 3 TVTVDKSX 4 RIVYMX 5 LSSLX 6 SEDX 7 AVYFCAR (SEQ ID NO: 48), wherein X 1 may be Q or K, X 2 may be D or G, X 3 may be A or V, X 4 may be S or T, X 5 may be E or Q, X 6 may be R or T, and X 7 may be S or T.
  • the H-FR3 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 24.
  • the H-FR4 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 49.
  • WGX 1 GTX 2 VTVSS (SEQ ID NO: 49), wherein X 1 may be Q or T and X 2 may be M or T.
  • the H-FR4 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 25.
  • the H-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 22; the H-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 23; the H-FR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 24; and the H-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 25.
  • the H-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 5; the H-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 6; the H-FR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 7; and the H-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO: 8.
  • the reference antibody may comprise Antibody 8H2D7B3 or an antibody having the same H-FR1-4 as Antibody 8H2D7B3.
  • the H-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 5; the H-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 6; the H-FR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 7; and the H-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO: 8.
  • the reference antibody may include Antibody JYB1907hz0 or an antibody having the same H-FR1-4 as Antibody JYB1907hz0.
  • the H-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 22; the H-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 23; the H-FR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 24; and the H-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO: 25.
  • the reference antibody may include Antibody JYB1907hz18 or an antibody having the same H-FR1-4 as Antibody JYB1907hz18.
  • the reference antibody may comprise a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 50.
  • X 1 may be Q or V
  • X 2 may be L or V
  • X 3 may be K or V
  • X 4 may be K or R
  • X 5 may be A or S
  • X 6 may be L or V
  • X 7 may be K or R
  • X 8 may be A or R
  • X 9 may be G or I
  • X 10 may be Q or K
  • X 11 may be D or G
  • X 12 may be A or V
  • X 13 may be S or T
  • X 14 may be E or Q
  • X 15 may be
  • the heavy chain variable region of the reference antibody may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 1 and SEQ ID NO: 21.
  • the reference antibody may comprise a heavy chain constant region, which may comprise an IgG-derived constant region or an IgY-derived constant region.
  • the heavy chain constant region of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 17.
  • the reference antibody may comprise a light chain variable region VL, which may comprise LCDR1, LCDR2, and LCDR3.
  • the HCDR3 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the LCDR2 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 45.
  • GATSLEX 1 (SEQ ID NO: 45), wherein, X 1 may be S or T.
  • the LCDR2 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 11 or SEQ ID NO: 28.
  • the LCDR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 44.
  • X 1 ASDHINKWLA (SEQ ID NO: 44), wherein, X 1 may be K or R.
  • the LCDR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 27.
  • the reference antibody may comprise Antibody 8H2D7B3 or an antibody having the same LCDR1-3 as Antibody 8H2D7B3.
  • the reference antibody may include Antibody JYB1907hz0 or an antibody having the same LCDR1-3 as Antibody JYB 1907hz0.
  • the reference antibody may include Antibody JYB1907hz18 or an antibody having the same LCDR1-3 as Antibody JYB1907hz18.
  • the VL of the reference antibody may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the L-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 52.
  • X 1 may be P or S
  • X 2 may be A or S
  • X 3 may be T or Y
  • X 4 may be A or V
  • X 5 may be L or V
  • X 6 may be D or G.
  • the L-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 29.
  • the L-FR2 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 14
  • the L-FR3 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 53.
  • X 1 PSRFSGSGSGKDYTLX 2 IX 3 X 4 LQX 5 X 6 DX 7 ATYYC (SEQ ID NO: 53), wherein, X 1 maybe I or V, X 2 may be TorS, X 3 may be TorS, X 4 may be G or S, X 5 may be P or T, X 6 may be D or E, X 7 may be F or V
  • the L-FR3 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 15 or SEQ ID NO: 30.
  • the L-FR4 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 54.
  • FGX 1 GTKLEIK (SEQ ID NO: 54), wherein, X 1 may be G or Q.
  • the L-FR4 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 16 or SEQ ID NO: 31.
  • the L-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 13; the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14; the L-FR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 15; and the L-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO: 16.
  • the reference antibody may comprise Antibody 8H2D7B3 or an antibody having the same L-FR1-4 as the antibody8H2D7B3.
  • the L-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 13; the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14; the L-FR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 15; and the L-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO: 16.
  • the reference antibody may include Antibody JYB1907hz0 or an antibody having the same H-FR1-4 as Antibody JYB1907hz0.
  • the L-FR1 of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 29; the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14; the L-FR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 30; and the L-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO: 31.
  • the reference antibody may include Antibody JYB1907hz18 or an antibody having the same L-FR1-4 as Antibody JYB 1907hz18.
  • the reference antibody may comprise a light chain variable region, which may comprise an amino acid sequence as set forth in SEQ ID NO: 51.
  • X 1 may be P or S
  • X 2 may be A or S
  • X 3 may be T or Y
  • X 4 may be A or V
  • X 5 may be L or V
  • X 6 may be D or G
  • X 7 may be K or R
  • X 8 may be SorT
  • X 9 may be I or V
  • X 10 may be T or S
  • X 11 may be T or S
  • X 12 may be G or S
  • X 13 may be P or T
  • X 14 may be D or E
  • X 15 may be F or V
  • X 16 may be G or Q.
  • the light chain variable region of the reference antibody may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 9 and SEQ ID NO: 26.
  • the reference antibody may comprise a light chain constant region, which may comprise an Ig ⁇ -derived constant region or an Ig ⁇ -derived constant region.
  • the reference antibody light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the reference antibody may comprise Antibody 8H2D7B3 or an antigen binding protein having the same HCDR1-3 and LCDR1-3 as Antibody 8H2D7B3.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the reference antibody may include Antibody JYB1907hz0 or an antigen binding protein having the same HCDR1-3 and LCDR1-3 as Antibody JYB 1907hz0.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 27; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 28; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the reference antibody may include Antibody JYB1907hz18 or an antigen binding protein having the same HCDR1-3 and LCDR1-3 as Antibody JYB1907hz18.
  • the reference antibody may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4;
  • the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10;
  • the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the L-FR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 13;
  • the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14;
  • the heavy chain variable region of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 1.
  • the reference antibody may comprise an antigen-binding fragment 8H2D7B3 or an antigen-binding protein having the same heavy chain variable region as the antigen-binding fragment 8H2D7B3.
  • the light chain variable region of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 9.
  • the reference antibody may comprise Antibody 8H2D7B3 or an antigen-binding protein having the same light chain variable region as Antibody 8H2D7B3.
  • the reference antibody comprises a heavy chain and a light chain
  • the heavy chain of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 19.
  • the reference antibody may comprise Antibody 8H2D7B3 or an antigen-binding protein having the same heavy chain as Antibody 8H2D7B3.
  • the light chain of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 20.
  • the reference antibody may include Antibody 8H2D7B3 or an antigen-binding protein having the same light chain as Antibody 8H2D7B3.
  • the reference antibody may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4;
  • the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10;
  • the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the L-FR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 13;
  • the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14;
  • the heavy chain variable region of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 1.
  • the reference antibody may comprise an antigen-binding fragment JYB1907hz0 or an antigen-binding protein having the same heavy chain variable region as the antigen-binding fragment JYB1907hz0.
  • the light chain variable region of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 9.
  • the reference antibody may include Antibody JYB 1907hz0 or an antigen-binding protein having the same light chain variable region as Antibody JYB 1907hz0.
  • the reference antibody may comprise a heavy chain and a light chain
  • the heavy chain of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 19.
  • the reference antibody may include Antibody JYB1907hz0 or an antigen-binding protein having the same heavy chain as Antibody JYB1907hz0.
  • the light chain of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 20.
  • the reference antibody may include Antibody JYB1907hz0 or an antigen-binding protein having the same light chain as Antibody JYB 1907hz0.
  • the reference antibody may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4;
  • the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 27;
  • the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 28; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the L-FR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 29;
  • the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14;
  • the heavy chain variable region of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 21.
  • the reference antibody may comprise an antigen-binding fragment JYB1907hz18 or an antigen-binding protein having the same heavy chain variable region as the antigen-binding fragment JYB1907hz18.
  • the light chain variable region of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 26.
  • the reference antibody may include Antibody JYB1907hz18 or an antigen-binding protein having the same light chain variable region as Antibody JYB1907hz18.
  • the reference antibody may comprise a heavy chain and a light chain.
  • the heavy chain of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 32.
  • the reference antibody may include Antibody JYB1907hz18 or an antigen-binding protein having the same heavy chain as Antibody JYB1907hz18.
  • the light chain of the reference antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 33.
  • the reference antibody may include Antibody JYB1907hz18 or an antigen-binding protein having the same light chain as Antibody JYB1907hz18.
  • the isolated antigen-binding protein may bind to the human GARP/human TGF- ⁇ 1 complex.
  • the isolated antigen-binding protein described herein may remit or treat a tumor, wherein the tumor may include a solid tumor.
  • tumors associated with the expression of GARP may be included.
  • the tumor may be a metastatic colon cancer.
  • the tumor may be a hepatocellular carcinoma.
  • the tumor may be an advanced renal cell carcinoma.
  • the tumor may be a non-small cell lung cancer.
  • the tumor may be a melanoma, a breast tumor, and/or a lung tumor.
  • the tumor may be a melanoma, a breast cancer and/or a lung squamous carcinoma.
  • the tumor may be a melanoma.
  • the isolated antigen-binding protein may comprise a heavy chain variable region VH which may comprise at least one, at least two, or at least three of HCDR1, HCDR2, and HCDR3.
  • the HCDR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 4.
  • the HCDR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 3.
  • the HCDR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 2.
  • the HCDR1 of an isolated antigen-binding protein described herein may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antibody having the same HCDR1-3 as Antibody 8H2D7B3.
  • the HCDR1 of an isolated antigen-binding protein described herein may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antibody having the same HCDR1-3 as Antibody JYB 1907hz0.
  • the HCDR1 of an isolated antigen-binding protein described herein may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4.
  • the isolated antigen-binding protein may include Antibody JYB1907hz18 or an antibody having the same HCDR1-3 as Antibody JYB1907hz18.
  • the VH of the antigen-binding protein may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the H-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 46.
  • X 1 may be Q or V
  • X 2 may be L or V
  • X 3 may be K or V
  • X 4 may be K or R
  • X 5 may be A or S
  • X 6 may be L or V
  • the H-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 22.
  • the H-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 47.
  • WMYWVX 1 QX 2 PX 3 QGLEWIGSI (SEQ ID NO: 47), wherein X 1 may be K or R, X 2 may be A or R, and X 3 may be G or I.
  • the H-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 23.
  • the H-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 48.
  • THYNQKFX 1 X 2 RX 3 TVTVDKSX 4 RIVYMX 5 LSSLX 6 SEDX 7 AVYFCAR (SEQ ID NO: 48), wherein X 1 may be Q or K, X 2 may be D or G, X 3 may be A or V, X 4 may be S or T, X 5 may be E or Q, X 6 may be R or T, and X 7 may be S or T.
  • the H-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 24.
  • the H-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 49.
  • WGX 1 GTX 2 VTVSS (SEQ ID NO: 49)
  • X 1 may be Q or T
  • X 2 may be M or T.
  • the H-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 25.
  • the H-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 22; the H-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 23; the H-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 24; and the H-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 25.
  • the H-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 5; the H-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 6; the H-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 7; and the H-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 8.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antibody having the same H-FR1-4 as Antibody 8H2D7B3.
  • the H-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 5; the H-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 6; the H-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 7; and the H-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 8.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antibody having the same H-FR1-4 as Antibody JYB 1907hz0.
  • the H-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 22; the H-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 23; the H-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 24; and the H-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 25.
  • the isolated antigen-binding protein may include Antibody JYB 1907hz 18 or an antibody having the same H-FR1-4 as Antibody JYB 1907hz18.
  • the isolated antigen-binding protein may comprise a heavy chain variable region, which may comprise an amino acid sequence as set forth in SEQ ID NO: 50.
  • X 1 may be Q or V
  • X 2 may be L or V
  • X 3 may be K or V
  • X 4 may be K or R
  • X 5 may be A or S
  • X 6 may be L or V
  • X 7 may be K or R
  • X 8 may be A or R
  • X 9 may be G or I
  • X 10 may be Q or K
  • X 11 may be D or G
  • X 12 may be A or V
  • X 13 may be S or T
  • X 14 may be E or Q
  • X 15 may be
  • the antigen-binding protein heavy chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 21.
  • the isolated antigen-binding protein may comprise a heavy chain constant region, which may comprise an IgG-derived constant region or an IgY-derived constant region.
  • the heavy chain constant region may comprise an IgG1-derived or IgG4-derived constant region.
  • the antigen-binding protein light chain constant region may comprise an amino acid sequence as set forth in SEQ ID NO: 17.
  • the isolated antigen-binding protein may comprise a light chain variable region VL, which may comprise LCDR1, LCDR2, and LCDR3.
  • the LCDR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the LCDR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 45.
  • GATSLEX 1 (SEQ ID NO: 45), wherein, X 1 may be S or T.
  • the LCDR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 11 or SEQ ID NO: 28.
  • the LCDR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 44.
  • X 1 ASDHINKWLA (SEQ ID NO: 44), wherein, X 1 may be K or R.
  • the LCDR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 27.
  • the LCDR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antibody having the same LCDR1-3 as Antibody 8H2D7B3.
  • the LCDR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antibody having the same LCDR1-3 as Antibody JYB1907hz0.
  • the LCDR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 27; the LCDR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 28; and the LCDR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein may include Antibody JYB1907hz18 or an antibody having the same LCDR1-3 as Antibody JYB1907hz18.
  • the VL of the antigen-binding protein may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the L-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 52.
  • X 1 may be P or S
  • X 2 may be A or S
  • X 3 may be T or Y
  • X 4 may be A or V
  • X 5 may be L or V
  • X 6 may be D or G.
  • the L-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 29.
  • the L-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 14.
  • the L-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 53.
  • X 1 PSRFSGSGSGKDYTLX 2 IX 3 X 4 LQX 5 X 6 DX 7 ATYYC (SEQ ID NO: 53), wherein, X 1 may be I or V, X 2 may be T or S, X 3 may be T or S, X 4 may be G or S, X 5 may be P or T, X 6 may be D or E, X 7 may be F or V
  • the L-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 15 or SEQ ID NO: 30.
  • the L-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 54.
  • FGX 1 GTKLEIK (SEQ ID NO: 54), wherein, X 1 may be G or Q.
  • the L-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 16 or SEQ ID NO: 31.
  • the L-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 15 or SEQ ID NO: 30;
  • the L-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 16 or SEQ ID NO: 31.
  • the L-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 13; the L-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 14; the L-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 15; and the L-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 16.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antibody having the same L-FR1-4 as Antibody 8H2D7B3.
  • the L-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 13; the L-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 14; the L-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 15; and the L-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 16.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antibody having the same H-FR1-4 as Antibody JYB 1907hz0.
  • the L-FR1 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 29; the L-FR2 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 14; the L-FR3 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 30; and the L-FR4 of the antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 31.
  • the isolated antigen-binding protein may include Antibody JYB1907hz18 or an antibody having the same L-FR1-4 as Antibody JYB 1907hz18.
  • the isolated antigen-binding protein may comprise a light chain variable region, which may comprise an amino acid sequence as set forth in SEQ ID NO: 51.
  • X 1 may be P or S
  • X 2 may be A or S
  • X 3 may be T or Y
  • X 4 may be A or V
  • X 5 may be L or V
  • X 6 may be D or G
  • X 7 may be K or R
  • X 8 may be SorT
  • X 9 may be I or V
  • X 10 may be T or S
  • X 11 may be T or S
  • X 12 may be G or S
  • X 13 may be P or T
  • X 14 may be D or E
  • X 15 may be F or V
  • X 16 may be G or Q.
  • the antigen-binding protein light chain variable region may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 9 and SEQ ID NO: 26.
  • the isolated antigen-binding protein may comprise a light chain constant region, which comprises an Ig ⁇ -derived constant region or an Ig ⁇ -derived constant region.
  • the antigen-binding protein light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18.
  • the isolated antigen-binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antigen-binding protein having the same HCDR1-3 and LCDR1-3 as Antibody 8H2D7B3.
  • the isolated antigen-binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antigen-binding protein having the same HCDR1-3 and LCDR1-3 as Antibody JYB1907hz0.
  • the isolated antigen-binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 27; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 28; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the isolated antigen-binding protein may include Antibody JYB1907hz18 or an antigen-binding protein having the same HCDR1-3 and LCDR1-3 as Antibody JYB1907hz18.
  • the isolated antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the L-FR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 13;
  • the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14;
  • the isolated antigen-binding fragment heavy chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 1.
  • the isolated antigen-binding protein may comprise an antigen-binding fragment 8H2D7B3 or an antigen-binding protein having the same heavy chain variable region as the antigen-binding fragment 8H2D7B3.
  • the isolated antigen-binding protein light chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 9.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antigen-binding protein having the same light chain variable region as Antibody 8H2D7B3.
  • the isolated antigen-binding protein may comprise a heavy chain and a light chain.
  • the heavy chain of the isolated antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 19.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antigen-binding protein having the same heavy chain as Antibody 8H2D7B3.
  • the light chain of the isolated antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 20.
  • the isolated antigen-binding protein may comprise Antibody 8H2D7B3 or an antigen-binding protein having the same light chain as Antibody 8H2D7B3.
  • the isolated antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 11; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the L-FR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 13;
  • the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14;
  • the heavy chain variable region of the isolated antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 1.
  • the isolated antigen-binding protein may comprise an antigen-binding fragment JYB1907hz0 or an antigen-binding protein having the same heavy chain variable region as the antigen-binding fragment JYB1907hz0.
  • the isolated antigen-binding protein light chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 9.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antigen-binding protein having the same light chain variable region as Antibody JYB 1907hz0.
  • the isolated antigen-binding protein may comprise a heavy chain and a light chain.
  • the heavy chain of the isolated antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 19.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antigen-binding protein having the same heavy chain as Antibody JYB1907hz0.
  • the light chain of the isolated antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 20.
  • the isolated antigen-binding protein may include Antibody JYB1907hz0 or an antigen-binding protein having the same light chain as Antibody JYB 1907hz0.
  • the isolated antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 2; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 3; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 27; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 28; and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 12.
  • the L-FR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 29;
  • the L-FR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 14;
  • the isolated antigen-binding protein heavy chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 21.
  • the isolated antigen-binding protein may include an antigen-binding fragment JYB1907hz18 or an antigen-binding protein having the same heavy chain variable region as the antigen-binding fragment JYB1907hz18.
  • the isolated antigen-binding protein light chain variable region may comprise an amino acid sequence as set forth in SEQ ID NO: 26.
  • the isolated antigen-binding protein may include Antibody JYB1907hz18 or an antigen-binding protein having the same light chain variable region as Antibody JYB1907hz18.
  • the isolated antigen-binding protein may comprise a heavy chain and a light chain.
  • the heavy chain of the isolated antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 32.
  • the isolated antigen-binding protein may include Antibody JYB1907hz18 or an antigen-binding protein having the same heavy chain as Antibody JYB1907hz18.
  • the light chain of the isolated antigen-binding protein may comprise an amino acid sequence as set forth in SEQ ID NO: 33.
  • the isolated antigen-binding protein may include Antibody JYB 1907hz 18 or an antigen-binding protein having the same light chain as Antibody JYB1907hz18.
  • the present application provides one or more polypeptides, which may comprise an isolated antigen-binding protein of the present application.
  • the present application provides one or more immunoconjugates, which may comprise an isolated antigen-binding protein of the present application.
  • the immunoconjugate also comprises a pharmaceutically acceptable therapeutic agent.
  • the therapeutic agent may be a cytotoxic agent or a cytostatic agent.
  • the therapeutic agent may be selected from the group consisting of: mitotic inhibitors, kinase inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylase inhibitors, anti-survival agents, and biological response modifiers.
  • the present application also provides one or more isolated nucleic acid molecules that may encode the isolated antigen-binding proteins described herein.
  • each of the one or more nucleic acid molecules may encode the entire antigen-binding protein or a portion thereof (e.g., one or more of the HCDR1-3 and the heavy chain variable regions).
  • the nucleic acid molecules described herein may be isolated. For example, it may be produced or synthesized by the following methods: (i) in vitro amplification, e.g., by polymerase chain reaction (PCR) amplification, (ii) clonal recombination, (iii) purification, e.g., by digestion and gel electrophoresis fractionation, or (iv) synthesis, e.g., by chemical synthesis.
  • the isolated nucleic acid may be a nucleic acid molecule prepared by recombinant DNA technology.
  • nucleic acids encoding the isolated antigen-binding proteins may be prepared by a variety of methods known in the art including, but not limited to, using reverse transcription PCR and PCR to obtain nucleic acid molecules of the isolated antigen-binding proteins described herein.
  • the present application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • Each vector may comprise one or more of the nucleic acid molecules.
  • the vector may also comprise other genes, such as marker genes that allow for selection of the vector in an appropriate host cell and under appropriate conditions.
  • the vector may also comprise expression control elements that allow for the proper expression of the coding region in an appropriate host.
  • control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, etc.
  • the expression control sequence is a regulatable element.
  • the specific structure of the expression control sequences may vary depending on the function of the species or cell type, but typically comprise 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA cassettes, capping sequences, CAAT sequences, etc.
  • the 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcription control of a functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6RNA promoters, CMV promoters, and artificial hybrid promoters thereof (e.g., CMV), wherein some portion of the promoter may be fused to some portion of the promoter of a gene for another cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not comprise an additional intron.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control elements.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector may be an expression vector.
  • the vector may be a viral vector.
  • the patient may be administered directly (in vivo) with the viral vector or may be administered indirectly, e.g., the patient may be administered with the cell treated with the virus in vitro (ex vivo).
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al., (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York ) and other virology and molecular biology manuals.
  • Retroviral vectors may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors for gene transfer.
  • retroviral, lentiviral, and adeno-associated viral methods can be used to transfer and integrate gene into the host genome for long term expression of the inserted gene.
  • Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers.
  • Lentiviral vectors may comprise a long terminal repeat 5' LTR and a truncated 3' LTR, a RRE, a rev response element (cPPT), a central termination sequence (CTS), and/or a post-translational regulatory element (WPRE).
  • the vectors described herein can be introduced into cells.
  • the present application provides a cell.
  • the cell may comprise an isolated antigen-binding protein as described herein, a polypeptide as described herein, an immunoconjugate as described herein, one or more nucleic acid molecules and/or one or more vectors as described herein.
  • each or every cell may comprise a nucleic acid molecule or vector described herein.
  • each or every cell may comprise many (e.g., 2 or more) or multiple (e.g., 2 or more) kinds of nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into said host cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS/0 cells, HEK293T cells, 293F cells, or HEK293A cells, or other eukaryotic cells, such as cells from plants, fungal or yeast cells, etc.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.
  • the cells may include yeast cells.
  • the cells may include E. coli cells.
  • the cells may include mammalian cells.
  • the cells may include immune cells.
  • the cells may include immune cells.
  • the cells may include immune cells.
  • the cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells.
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may comprise the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or the pharmaceutically acceptable adjuvant and/or excipient as described herein.
  • the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and/or nonionic surfactants. Any conventional media or agent is contemplated for the pharmaceutical compositions of the present application, unless they are incompatible with the cells described herein.
  • the pharmaceutically acceptable excipients may include an additive other than the main drug in the pharmaceutical preparation, which may also be referred to as pharmaceutical necessities.
  • the excipients may include binders, fillers, disintegrants, and lubricants in tablets.
  • the excipients may include alcohol, vinegar, medicinal juice, etc. in traditional Chinese medicine pills.
  • the excipients may include the base portion of semi-solid formulation ointments and creams.
  • the excipients may include preservatives, antioxidants, flavoring agents, perfuming agents, solubilizing assistant, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid formulations.
  • the present application provides a pharmaceutical combination comprising the isolated antigen-binding protein and an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor may include a substance that inhibits the interaction of PD-1/PD-L1.
  • the immune checkpoint inhibitor is selected from the group consisting of: PD-1/PD-L1 blocking agents, PD-1 antagonists, PD-L1 antagonists, PD-1 inhibitors, and PD-L1 inhibitors.
  • the PD-1/PD-L1 blocking agent may be selected from the group consisting of: BMS202 (PD-1/PD-L1 inhibitor 2), BMS-1 (PD-1/PD-L1 inhibitor 1), PD-1/PD-L1 inhibitor 3, BMS-1166 and BMS-1001.
  • the PD-1 inhibitor may include an anti-PD-1 antibody.
  • the PD-L1 inhibitor may include a PD-L1 antibody.
  • the anti-PD-1 antibody may be selected from the group consisting of: Nivolumab, Pembrolizumab, Camrelizumab, Toripalimab, Sintilimab, and Tislelizumab.
  • the anti-PD-L1 antibody may be selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise HCDR3 of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise HCDR2 of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise HCDR1 of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise LCDR3 of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise LCDR2 of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise LCDR1 of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise a VH of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise a VL of an antibody selected from the group consisting of: Durvalumab, Atezolizumab, and avelumab.
  • the anti-PD-L1 antibody may comprise HCDR3, which may comprise an amino acid sequence as set forth in SEQ ID NO: 37.
  • the anti-PD-L1 antibody comprises HCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 36.
  • the anti-PD-L1 antibody comprises HCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 35.
  • the anti-PD-L1 antibody comprises a heavy chain variable region VH which comprises HCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 35, HCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 36, and HCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 37.
  • the antibody may include Atezolizumab or an antibody having the same HCDR1-3 as the Atezolizumab.
  • the anti-PD-L1 antibody may comprise a heavy chain variable region VH, which may comprise an amino acid sequence as set forth in SEQ ID NO: 34.
  • the anti-PD-L1 antibody may comprise LCDR3, which may comprise an amino acid sequence as set forth in SEQ ID NO: 41.
  • the anti-PD-L1 antibody may comprise LCDR2, which may comprise an amino acid sequence as set forth in SEQ ID NO: 40.
  • the anti-PD-L1 antibody may comprise LCDR1, which may comprise an amino acid sequence as set forth in SEQ ID NO: 39.
  • the anti-PD-L1 antibody may comprise a light chain variable region VL, which may comprise LCDR1, LCDR2, and LCDR3, wherein the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 41; the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 40; and the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 39.
  • the antibody may include Atezolizumab or an antibody having the same LCDR1-3 as the Atezolizumab.
  • the anti-PD-L1 antibody may comprise a light chain variable region VL, which may comprise an amino acid sequence as set forth in SEQ ID NO: 38.
  • the anti-PD-L1 antibody may comprise a heavy chain and a light chain, wherein the heavy chain may comprise HCDR1-3 and H-FR1-4, and the light chain may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 35; the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 36; and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 37;
  • the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 39;
  • the anti-PD-L1 antibody may include Atezolizumab or an antigen-binding protein having the same HCDR1-3 and LCDR1-3 as the Atezolizumab.
  • the anti-PD-L1 antibody heavy chain variable region can comprise an amino acid sequence as set forth in SEQ ID NO: 34.
  • the anti-PD-L1 antibody may include Atezolizumab or an antigen-binding protein having the same heavy chain variable region as the Atezolizumab.
  • the light chain variable region of the anti-PD-L1 antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 38.
  • the anti-PD-L1 antibody may include Atezolizumab or an antigen-binding protein having the same light chain variable region as the Atezolizumab.
  • the heavy chain of the anti-PD-L1 antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 42.
  • the anti-PD-L1 antibody may include Atezolizumab or an antigen-binding protein having the same heavy chain as the Atezolizumab.
  • the light chain of the anti-PD-L1 antibody may comprise an amino acid sequence as set forth in SEQ ID NO: 43.
  • the anti-PD-L1 antibody may include Atezolizumab or an antigen-binding protein having the same light chain as the Atezolizumab.
  • the present application provides a kit comprising the pharmaceutical combination as described herein.
  • the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and the pharmaceutical composition for the prevention, remission, and/or treatment of a tumor.
  • the tumor may include a solid tumor.
  • the tumor may include a tumor associated with protein expression of GARP.
  • the tumor may be a metastatic colon cancer.
  • the tumor may be a hepatocellular carcinoma.
  • the tumor may be an advanced renal cell carcinoma.
  • the tumor may be a non-small cell lung cancer.
  • the tumor may be a melanoma, a breast tumor, and/or a lung tumor.
  • the kit and/or the pharmaceutical combination is used for the prevention, remission, and/or treatment of a tumor.
  • the tumor may include a solid tumor.
  • the tumor may include a tumor associated with protein expression of GARP.
  • the tumor may be a metastatic colon cancer.
  • the tumor may be a hepatocellular carcinoma.
  • the tumor may be an advanced renal cell carcinoma.
  • the tumor may be a non-small cell lung cancer.
  • the tumor may be a melanoma, a breast tumor, and/or a lung tumor.
  • the present application provides a use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition in the manufacture of a medicament for the prevention, remission, and/or treatment of a tumor.
  • the tumor may include a solid tumor.
  • the tumor may include a tumor associated with protein expression of GARP.
  • the tumor may be a metastatic colon cancer.
  • the tumor may be a hepatocellular carcinoma.
  • the tumor may be an advanced renal cell carcinoma.
  • the tumor may be a non-small cell lung cancer.
  • the tumor may be a melanoma, a breast tumor, and/or a lung tumor.
  • the present application provides a use of a pharmaceutical combination and/or a kit in the manufacture of a medicament for the prevention, remission, and/or treatment of a tumor.
  • the tumor may include a solid tumor.
  • the tumor may include a tumor associated with protein expression of GARP.
  • the tumor may be a metastatic colon cancer.
  • the tumor may be a hepatocellular carcinoma.
  • the tumor may be an advanced renal cell carcinoma.
  • the tumor may be a non-small cell lung cancer.
  • the tumor may be a melanoma, a breast tumor, and/or a lung tumor.
  • the present application provides a method for the prevention and/or treatment of a disease or disorder comprising administering to a subject in need thereof the isolated antigen-binding protein, the isolated nucleic acid molecule, the vector, the cell, and the pharmaceutical composition, wherein the disease or disorder includes a tumor.
  • the present application provides a method for the prevention and/or treatment of a disease or disorder comprising administering to a subject in need thereof the pharmaceutical combination, wherein the disease or disorder includes a tumor.
  • the tumor may include a solid tumor.
  • the tumor may include a tumor associated with protein expression of GARP.
  • the tumor may be a metastatic colon cancer.
  • the tumor may be a hepatocellular carcinoma.
  • the tumor may be an advanced renal cell carcinoma.
  • the tumor may be a non-small cell lung cancer.
  • the tumor may be a melanoma, a breast tumor, and/or a lung tumor.
  • the pharmaceutical composition, pharmaceutical combination, and method described herein can be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, and gene therapy, etc.
  • the pharmaceutical composition and method described herein can be used for other disease conditions that depend on an immune response, such as inflammation, immune diseases, and infectious diseases.
  • the subject may include a human or non-human animal.
  • the non-human animal may be selected from the group consisting of: a monkey, a chicken, a goose, a cat, a dog, a mouse, and a rat.
  • a non-human animal may also include any animal species other than human, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
  • the human can be Caucasian, African, Asian, Sumerian, or other ethnicity, or a hybrid of various ethnicities.
  • the human may be the elderly, adults, adolescents, children or infants.
  • Effective amounts in human can be extrapolated from the effective amounts in experimental animals.
  • Freireich et al. described the interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) ( Freireich et al., Cancer Chemother. Rep. 50, 219 (1966 )).
  • Body surface area can be approximately determined from the height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y, 537 (1970 ).
  • the full-length amino acid sequence of human GARP (designated as hGARP) (Uniprot#Q14392) and the full-length amino acid sequence of human Latent TGF- ⁇ (designated as hTGF- ⁇ 1) (Uniprot#P01137) were codon-optimized to synthesize DNA sequences, which were cloned into the vectors pLVX-IRES.puro and pLVX-IRES.G418, respectively, according to the established molecular biological methods; the constructed recombinant plasmids were packaged by lentivirus, then infected into 293F cells, and screened by antibiotic resistance; the 293F-hGARP/hTGF- ⁇ 1 monoclonal recombinant cell strain over-expressing hGARP/hTGF- ⁇ 1 was obtained by picking a monoclonal colony, stained with flow anti-human GARP antibody (Enzo life science, ALX-804-867FI-0100) and anti-human TGF- ⁇ 1 antibody (R
  • mice were immunized with 293F-hGARP/hTGF- ⁇ 1 monoclonal recombinant cell strains 2F4 and 2D3 for 3-4 times, respectively, 3-5 ⁇ 10 6 cells/mouse were injected at 2-3 weeks intervals, and blood was collected on the seventh day after the third immunization as post-immunization serum.
  • the binding degree of the immunized serum was detected with CHOK1-hGARP/hTGF- ⁇ 1 and blank cell CHOK1 in flow cytometry, the ratio of the average fluorescence intensity for binding to the CHOK1-hGARP/hTGF- ⁇ 1 and the blank cell CHOK1 was calculated in the flow cytometry, and the mice with higher ratio were used for fusion.
  • mice were subjected to booster immunization with 293F-hGARP/hTGF- ⁇ 1 monoclonal recombinant cell strain at 3-5 ⁇ 10 6 cells/mouse.
  • mice On the day of fusion, after euthanasia of mice, the mice were dissected, the spleens were fetched and ground to harvest cells, which were collected by centrifugation at 1500 rpm and 5 min. The cells were suspended with 5 ml of red blood cell lysate, leaving standing at 4°C for 5 min, reactions were terminated with DMEM+10% FBS and the cells were counted. After centrifugation, the cells were suspended with 40 ml DMEM. After standing for 2-3 min, the supernatant was transferred to another 50 mL centrifuge tube.
  • Hybridoma supernatants from the 96-well cell culture plate were assayed for binding activity to CHOK1-hGARP/hTGF- ⁇ 1 cells by flow cytometry.
  • the CHOK1-hGARP/hTGF- ⁇ 1 monoclonal recombinant cell strain was expanded to 90% confluency in T-75 cell culture flasks, the culture medium was pipetted thoroughly, the culture was washed twice with PBS buffer (available from Invitrogen), then treated with enzyme-free cell dissociation buffer (Versene solution, 15040066, available from Life technology), and cells were collected. The cells were washed twice with PBS buffer, after cell counting, the cells were diluted to 2 ⁇ 10 6 cells/mL with PBS buffer, FACS buffer (PBS containing 2% FBS) was added, the mixture was incubated for 15 min at room temperature, and then centrifuged and washed twice with PBS buffer.
  • PBS buffer available from Invitrogen
  • enzyme-free cell dissociation buffer Versene solution, 15040066, available from Life technology
  • the collected cells were suspended to 3 ⁇ 10 6 cells/mL with FACS buffer.
  • the cell suspension was added to a 96 FACS reaction plate at 100 ⁇ L/well, the hybridoma supernatant from the 96-well cell culture plate was added at 100 ⁇ L/well, and the mixture was incubated at 4°C for 1 h. After centrifugation, the supernatant was discarded, a fluorescently (Alexa 488) labeled secondary antibody (avaiable from Invitrogen) was added at 100 ⁇ L/well, and the mixture was incubated for 1 h at 4°C.
  • the cells were centrifuged and washed 3 times with FACS buffer, suspended with the addition of fixative [4% (v/v) paraformaldehyde] at 100 ⁇ L/well, after 10 minutes, the cells were centrifuged and washed 2 times with FACS buffer. The cells were suspended with 30 ⁇ L of FACS buffer and the results were detected and analyzed with a flow cytometer intellycite plus (available from Sartorius).
  • the cell clone corresponding to the positive hybridoma supernatant was transferred to a 24-well cell culture plate for further cultivation for 2-4 days, and the hybridoma supernatant (corresponding cell clone name: 8H2) in the 24-well cell culture plate was used to assay binding activity to CHOK1-hGARP/hTGF- ⁇ 1 cells, and the results were shown in Table 2 below.
  • the positive cells were picked from the 24-well cell culture plate to perform subcloning and subcloning screening until obtaining a stable cell strain 8H2D7B3 capable of secreting a hybridoma monoclonal antibody which can binding to CHOK1-hGARP/hTGF- ⁇ 1, then the cell strain was cultured in a conventional serum-free medium for 50 ml of small-scale productiong, and purified with a conventional protein A column to obtain a purified monoclonal antibody for subsequent identification.
  • 5 ⁇ 10 7 hybridoma cells 8H2D7B3 were collected by centrifugation and total RNA was extracted by a Trizol method.
  • the cDNA obtained after the reverse transcription reaction was subjected to a G-addition reaction by terminal transferase, and the DNA containing the variable region sequence was amplified with VH, VK primers, and polyC primers, and T-A cloning was performed, followed by sequencing and antibody sequence analysis.
  • the cells were counted and centrifuged at 300 g for 5 min, the supernatant was discarded, the cell pellet was resuspended and washed with 1 ⁇ PBS, then centrifuged at 300 g for 5 min, the supernatant was discarded, and then the cell pellet was resuspended with FACS buffer containing 2% FBS to adjust to a density of 1e6 cells/mL.
  • the cells were plated at a density of 1e5 cells/well for a constant volume of 100 ⁇ L/well at 4°C for 0.5 h, and centrifuged at 300 g for 5 min, and the supernatant was discarded.
  • the antibody to be tested was prepared in FACS buffer containing 2% FBS with a starting concentration of 15 ⁇ g/mL, followed by serial dilution with a dilution factor of 1:5 in a total of 7 gradients, and then was added for a constant volume of 100 ⁇ L/well, with 0 ⁇ g/mL as a control, the dilution was pipetted and mixed thoroughly at 4°C for 1 h, and centrifuged at 300 g for 5 min, and the supernatant was discarded.
  • FIG. 1B shows the binding of the antibody to be tested to 293F-hGARP cells
  • Fig. 1C shows the binding of the antibody to be tested to 293F-hTGF- ⁇ 1 cells
  • Fig. 1D shows the binding of the antibody to be tested to 293F-hGARP/hTGF- ⁇ 1 complex, indicating that the murine antibody 8H2D7B3 binds only to the 293F-hGARP/hTGF- ⁇ 1 complex.
  • the cells were counted and centrifuged at 300 g for 5 min, the supernatant was discarded, the cell pellet was resuspended and washed with 1 ⁇ PBS, then centrifuged at 300 g for 5 min, the supernatant was discarded, and then the cell pellet was resuspended with FACS buffer containing 2% FBS to adjust to a density of 1e6 cells per mL.
  • the cells were plated at a density of 1e5 cells per well for a constant volume of 100 ⁇ L/well at 4°C for 0.5 h, and centrifuged at 300 g for 5 min, and the supernatant was discarded.
  • the competitor antibody with alexa488 (lot: 1905151702, 1.39 mg/mL) was added to a final concentration of 30 ⁇ g/mL for a constant volume of 50 ⁇ L/well; the antibody to be tested was prepared in FACS buffer containing 2% FBS with a starting concentration of 30 ⁇ g/mL, followed by serial dilution with a dilution factor of 1:5 in a total of 7 gradients, and then was added for a constant volume of 100 ⁇ L/well, with 0 ⁇ g/mL as a control, the mixture was pipetted and mixed thoroughly at 4°C for 1 h, and centrifuged at 300 g for 5 min, and the supernatant was discarded.
  • PBMC cells Fresh PBMC cells were purchased from ALLCELLS, firstly PBMC cells were centrifuged at 400 g for 10 min, then resuspended in 1 ⁇ PBS medium (Thermo Fisher, 10010049) containing 1% FBS (Thermo Fisher, 10099-141) and 50 mmol EDTA (Thermo Fisher, 15400054). PBMC was subjected to Treg cell extraction according to a Treg Extraction Kit (Miltenyi, Cat. No.: 130-091-301) protocol. Treg cell antibody treatment: Treg were expanded in vitro for 14 days, cells were collected and counted, and 1.5 ⁇ 10 6 Treg cells were depleted of magnetic beads and used as a negative control.
  • Treg cell antibody treatment Treg were expanded in vitro for 14 days, cells were collected and counted, and 1.5 ⁇ 10 6 Treg cells were depleted of magnetic beads and used as a negative control.
  • the cells were plated in a 24-well plate at 1 ⁇ 10 6 /mL per well for a constant volume of 1 mL/well.
  • the antibody to be tested was added to the 24-well plate at a concentration of 25 ⁇ g/mL.
  • the cells were cultured 36 h in a carbon dioxide incubator at 37°C.
  • Alpha Elisa assay the cells were collected by centrifugation, and 50 uL of Lysis buffer was added for lysis 30 min. The lysate was centrifuged at 1300 rpm for 5 min, and 10 uL supernatant was fetched for Alpha Elisa assay (PerkinElmer, ALSU-PSM2-A500).
  • Figs. 3A-3B Western blot assay: the cells were collected by centrifugation, and 50 uL of Lysis buffer was added for lysis 30 min. The lysate was centrifuged at 1300 rpm for 5 min, and 10 uL supernatant was fetched for Western blot assay. The results were shown in Figs. 3A-3B : Fig. 3A shows the Western blot assay results of the inhibition of SMAD2 phosphorylation in Treg cells by the antibody to be tested, and Fig.
  • 3B shows the Alpha Elisa assay signal value of the inhibition of SMAD2 phosphorylation in Treg cells by the antibody to be tested, indicating that the murine antibody was similar to the positive controls anti-TGF- ⁇ 1 and MHG8, both of which could inhibit the SMAD2 phosphorylation protein in Treg cells.
  • 8H2D7B3 was selected as murine candidate antibody and humanized.
  • variable region of the murine anti-human GARP monoclonal antibody 8H2D7B3 was determined as follows:
  • the CDR regions of the murine anti-human GARP monoclonal antibody 8H2D7B3 heavy and light chains were shown in Table 3.
  • Table 3 CDR regions of murine anti-human GARP monoclonal antibody 8H2D7B3 heavy and light chains Heavy chain Light chain HCDR1 GYTLSNY LCDR1 KASDHINKWLA HCDR2 APSDSE LCDR2 GATSLET HCDR3 GGFGYGSSHWYFDV LCDR3 QQYWTTPYT
  • the human Germline antibody (data source: IMGT) that is most homologous to the murine anti-human GARP monoclonal antibody 8H2D7B3 was selected by sequence alignment as a framework for humanization design (the light chain was framed with IGKV3D-15*01 or IGKV1-5*03, IGKJ2*02 or IGKJ4*02, and the heavy chain was framed with IGHV3-7*03 or IGHV1-46*03, IGHJ3*01), the CDR regions of the antibody were defined by Chothia numbering [Chothia & Lesk, 1987] of the variable regions of the antibody light and heavy chains: CDRL1(L24-L34), CDRL2(L50-L56), CDRL3(L89-L97), CDRH1(H26-H32), CDRH2(H52-H56), and CDRH3(H95-H97), amino acids in the variable region of the antibody light and heavy chains were subjected to humanization mutations
  • the expression vector is designed and the gene is synthesized. Then the recombinant antibodies are expressed in mammalian cells and purified.
  • the humanized antibody and chimeric antibody were compared in terms of activity and physicochemical properties, and 1-2 rounds of humanization optimization were performed, wherein the light and heavy chains are optimally designed by using the humanized sequence grafted by the Germinne antibody as a framework CDR.
  • the resulting chimeric antibody was named as JYB1907hz0 and the humanized antibody was named as JYB1907hz18.
  • the full-length JYB1907hz18 light and heavy chain protein sequence was codon-optimized, respectively, the codon-optimized DNA fragment (Genscript) was synthesized, and the synthesized gene fragment was cloned into expression vector pcDNA3.4 (Life Technologies).
  • expression plasmid amplification and plasmid extraction ExpiCHO cells (ThermoFisher Scientific, A29133) were co-transfected with two plasmids and transient antibody expression was performed according to the supplier's ExpiCHO expression system method.
  • ExpiCHO cells were cultured to a density of 6 ⁇ 10 6 /mL in a total culture volume of 25 ml culture medium at 36.5°C and 8% carbon dioxide concentration,. 10 ⁇ g of antibody light and heavy chain expression plasmids were respectively transferred to the cells using ExpiFectamine transfection reagent; one day after the transfection, 150 ⁇ L of ExpiCHO enhancer and 4 mL of ExpiCHO adjuvant was each added to the cultured cells, and the culture was continued for 9 days. The supernatant was obtained by centrifugation at 3500 rpm and 4°C.
  • AmMagTM Protein A magnetic beads (Genscript, L00695) and antibody expression supernatant were mixed, the mixture was incubated at room temperature for 2 h, and washed twice with PBS, the supernatant was discarded; an appropriate amount of elution buffer Protein G or A SefinoseTMElution buffer (Sangon, C600481) was added, the mixture was mixed thoroughly, and then placed on a test tube rack for static incubation for 5 min; the magnetic beads were resuspended for 2-3 times during the incubation, and the elution was repeated for 2 times. After elution, the elute was neutralized with an appropriate amount of neutralizing solution 1M Tris-HCl, pH7.5 (Sangon, B548124) immediately for later use .
  • Each cycle contains the following steps: 1) immersing the sample in buffer for 60 s; 2) detecting whether the antigen (hGARP/hTGF- ⁇ 1 complex) non-specifically binding to the sensor; 3) regenerating in a 1.0 mM glycine solution at pH 1.7; 4) immersing in buffer for 60 s; 5) immobilizing the antibody (JYB1907hz18) on the sensor for 25 s; 6) immersing the sensor in buffer solution for 180 s; 7) binding the antigen (hGARP/hTGF- ⁇ 1 complex) to the antibody (JYB1907hz18) for 180 s; 8) dissociating the antigen (hGARP/hTGF- ⁇ 1 complex) with antibody (JYB1907hz18) for 10 min; 9) regenerating the sensor.
  • the equilibrium dissociation constant (KD) of the antibody was calculated by measuring the association rate (Ka) and dissociation rate (Kd) of the antigen (hGARP/hTGF- ⁇ 1 complex)-antibody (JYB1907hz18) in a 1:1 binding mode using Data Analysis 12.0 software from Fortebio. Results were as shown in Table 4, JYB1907hz18 bound to hGARP/hTGF- ⁇ 1 with greater affinity than the positive control antibody MHG8.
  • PBMC cells Fresh PBMC cells were purchased from ALLCELLS, firstly PBMC cells were centrifuged at 400 g for 10 min, then resuspended in 1 ⁇ PBS medium (Thermo Fisher, 10010049) containing 1% FBS (Thermo Fisher, 10099-141) and 50 mmol EDTA (Thermo Fisher, 15400054). PBMC was subjected to Treg cell extraction according to a Treg Extraction Kit (Miltenyi, Cat. No.: 130-091-301) protocol. Treg cell antibody treatment: Treg were expanded in vitro for 14 days, cells were collected and counted, and 1.5 ⁇ 10 6 Treg cells were depleted of magnetic beads and used as a negative control.
  • Treg cell antibody treatment Treg were expanded in vitro for 14 days, cells were collected and counted, and 1.5 ⁇ 10 6 Treg cells were depleted of magnetic beads and used as a negative control.
  • the cells were plated in a 24-well plate at 1 ⁇ 10 6 /mL per well for a constant volume of 1 mL per well.
  • the antibody to be tested was added to the 24-well plate at a concentration of 25 ⁇ g/mL.
  • the cells were cultured 36 h in a carbon dioxide incubator at 37°C. Wherein, anti-TGF- ⁇ 1 and MHG8 were used as positive controls, and human IgG4 was used as an isotype control.
  • Alpha Elisa assay the cells were collected by centrifugation, and 50 uL of Lysis buffer was added for lysis 30 min. The lysate was centrifuged at 1300 rpm for 5 min, and 10 uL supernatant was fetched for Alpha Elisa assay (PerkinElmer, ALSU-PSM2-A500).
  • JYB 1907hz 18 inhibited SMAD2 phosphorylation in Treg cells as assessed by the method described above, with the results shown in Fig. 4 .
  • 293T-hGARP/hTGF- ⁇ 1 cells were constructed by Shanghai ChemPartner Co., Ltd., and LN229 cells were purchased from Nanjing Cobai (Cat. No.: CBP60302).
  • trypsin Thermo, 25200056
  • fresh DMEM Thermo Fisher, 11965092
  • FBS Thermo Fisher, 10099-141
  • Pen Strep Thermo Fisher, 15140-122
  • JYB1907hz18 inhibited the release of TGF- ⁇ 1 from 293T cells as assessed by the method described above, with the results shown in Fig. 5 .
  • Example 7 JYB1907hz18 preventing Treg from inhibiting the release of IL-2 and IFN ⁇ from human peripheral blood mononuclear cells (PBMC)
  • PBMC peripheral blood mononuclear cells
  • Fresh PBMC cells were purchased from TPCS, firstly PBMC cells were centrifuged at 400 g for 10 min, then resuspended in 1 ⁇ PBS medium (ThermoFisher, 10010049) containing 1% FBS (ThermoFisher, 10099-141) and 50 mmol EDTA (ThermoFisher, 15400054). A few PBMC were pipetted for subsequent experiments, and the remaining PBMCs were subjected to Treg cell extraction according to a Treg Extraction Kit (Miltenyi, Cat. No.: 130-091-301) protocol.
  • a Treg Extraction Kit Miltenyi, Cat. No.: 130-091-301
  • the extracted Treg cells and PBMC cells were counted and adjusted to a concentration of 5e5/mL, the PBMC group was supplemented with 50 ⁇ L of PBMC, 48 ⁇ L of TexMACS TM Medium medium (Miltenyi Biotec, 170-076-309) containing 1% HBS (TPCS, A515) and 1 ⁇ Pen Strep (ThermoFisher, 15140-122), and 2 ⁇ L of CD3/CD28 antibody magnetic beads (Miltenyi Biotec, 130-095-345).
  • the PBMC+Treg cell group was supplemented with 50 ⁇ L of PBMC, 46 ⁇ L of TexMACS TM Medium medium (Miltenyi Biotec, 170-076-309) containing 1% HBS (TPCS, A515) and 1 ⁇ Pen Strep (ThermoFisher, 15140-122), and 4 ⁇ L of CD3/CD28 antibody magnetic beads (Miltenyi Biotec, 130-095-345).
  • the antibody to be tested, JYB1907hz18 was added for a constant volume of 50 ⁇ L/well at a final concentration of 25 ⁇ g/ml. The mixture was incubated in a 37°C, 5% CO 2 incubator for 20-24 h.
  • the culture was centrifuged, and the supernatant was fetched and assayed for IL-2 (R&D, VAL110) and IFN- ⁇ (R&D, DIF50C) using IL-2 ELISA and IFN-y ELISA kits.
  • IL-2 R&D, VAL110
  • IFN- ⁇ R&D, DIF50C
  • MHG8 was used a positive control.
  • JYB1907hz18 inhibited the release of interleukin -2 (IL-2) and interferon- ⁇ (IFN- ⁇ ) from peripheral blood mononuclear cells (PBMCs) as assessed by the methods described above, with the results shown in Figs. 6A-6B: Fig. 6A shows that the antibody to be tested prevents Treg from inhibiting the release of interleukin-2 (IL-2) from PBMC; Fig. 6B shows that the antibody to be tested prevents Treg from inhibiting the release of interferon- ⁇ (IFN- ⁇ ) from PBMC.
  • IL-2 interleukin-2
  • IFN- ⁇ interferon- ⁇
  • Humanized FcRn mice were used as test animals, after single subcutaneous administration, the pharmacokinetic indexes of two drugs to be tested, MHG8 and JYB 1907hz 18, were studied, respectively. All animal experimental schemes were reviewed and approved by IACUC.
  • Male hFcRn mice aged between 6 and 8 weeks were purchased from Biocytogen Pharmaceuticals (Beijing) Co., Ltd., with body weight of 23-26 g, raised in SPF-grade animal room, fed with standard pellet feed, with free access to food and water at room temperature of 18-24°C and relative humidity of 40%-50%, exposed to daily alternating light-dark cycles (i.e., 12 h shifts). A total of 12 experimental animals were randomly divided into three groups, with four animals in each group.
  • the animals were subcutaneously administered with a single dose of 10 mg/kg at a dose volume of 10 mL/kg.
  • Blood was sampled before the drug administration, and 2 h, 6 h, 24 h (day 1), day 2, day 3, day 4, day 7, day 10, day 14, day 21, day 28, day 35, and day 42 after the drug administration.
  • Whole blood was collected into EP tubes at 60 ⁇ L/tube by puncturing cheek, allowed to stand at room temperature for 30 min, and then centrifuged (2000 g, 4°C, 5 min) to separate serum. Each sample was divided into 2 portions (a detection tube and a backup tube) at 10 ⁇ L/tube, and stored at -80°C.
  • Pharmacokinetics of three drugs at different time points were analyzed by indirect ELISA.
  • the plate was coated with the antigen, murine anti-human IgG4 Fc (Abcam, lot: GR3248093-2, ab99820) at 2 ⁇ g/mL, 100 ⁇ L/well, overnight at 4°C, then washed, and blocked with a blocking buffer at 200 ⁇ L/well overnight at 4°C. Serum sample was added at 37°C for 1 h, for a constant volume of 50 ⁇ l/well.
  • TMB developing solution KPL, Cat. No.: 52-00-03
  • OD450 was read on a microplate reader (Molecular Devices, SpectraMax M3).
  • Drug concentration was obtained according to a standard curve, and PK parameters were obtained by PK Solver non-compartmental data processing.
  • the sample (JYB1907hz18) was diluted with sample buffer to 1 mg/mL, then according to the instructions of Protein Thermal Shift TM Starter Kit, 13 ⁇ L of sample (JYB1907hz18) solution was added into a PCR tube, 5 ⁇ L of Protein Thermal shift TM Buffer was added, 2 ⁇ L of 10 ⁇ staining solution was added, resulting a reaction volume of 20 ⁇ L, the mixture was mixed thoroughly, and centrifuged at 12000 rpm for 5 min to remove air bubbles. The test sample (JYB1907hz18) was placed in the PCR instrument for sample analysis, and the Tm value of sample (JYB1907hz18) was recorded.
  • the sample (JYB1907hz18) solution was added into the following thoroughly mixed system: 70 ⁇ L of 1% methylcellulose (MC), 80 ⁇ L of 5M urea, 8 ⁇ L of amphoteric electrolyte Pharmalyte pH 3-10, 2 ⁇ L of pI marker 5.5, 2 ⁇ L of pI marker 9.5.
  • the system was supplemented with appropriate volume of ultrapure water to 200 ⁇ l, and the mixture was mixed thoroughly, then centrifuged, and the supernatant was analysized. After the analysis, the result file was imported into ChromPerfect software for spectrum integration processing and calculation of the isoelectric point of each peak as well as the percentage of each peak.
  • NOG mice 40 female NOG mice aged between 7 and 8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; Cryopreserved PBMC was purchased from Miaotong (Shanghai) Biotechnology Co., Ltd.; Treg cells were extracted from fresh PBMC and expanded in vitro as described in Example 5. After NOG mice were acclimated, mice were treated with 25 mg/kg of busulfan intraperitoneally 3 days in advance. Ten mice were randomly selected, and cryopreserved PBMCs from the same batch were recovered. Each mouse was intravenously injected with 3 ⁇ 10 6 PBMC as the first group.
  • mice were mixed with Treg cells, and the remaining 30 mice were injected intravenously with 3 ⁇ 10 6 PBMCs and 3 ⁇ 10 6 Treg cells via the tail vein. On the day after injection, the mice were randomly divided into groups 2, 3, and 4 according to body weight.
  • mice in the first group was intraperitoneally injected with isotype Ctrl
  • the mice in the second group was intraperitoneally injected with isotype Ctrl
  • the mice in the third group was intraperitoneally injected with MHG8 (positive control) at a dose of 20 mg/kg
  • the mice in the fourth group was intraperitoneally injected with JYB1907hz18 at a dose of 20 mg/kg, with administration frequency of twice a week, for 12 times in total.
  • Mice were weighed twice weekly and subjected to GvHD scoring, and survival was counted.
  • the GvHD scoring criteria were shown in Table 8. Mice were euthanized when they lost more than 25% of their body weight.
  • Table 8 GvHD scoring criteria Loss of weight -5% -15% -25% 1 score 2 scores 3 scores Jaundice or anemia 1 score Hollow back 1 score Range of motion Decreased range of motion Inactive 1 score 2 scores Hair loss/blowing 1 score Death 8 scores
  • Figs. 8A-8B show the results of Figs. 8A-8B: Fig. 8A shows the GvHD score of the antibody to be tested in mice; Fig. 8B shows the survival rate of the antibody to be tested in mice.
  • mice 48 female NOG-DKO mice aged between 7 and 8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; cryopreserved PBMC was purchased from Miaotong (Shanghai) Biotechnology Co., Ltd.; A375 human melanoma cells were purchased from ATCC.
  • the PBMC were recovered and PBMC was injected into each mouse via the tail vein in an amount of 6E6 to establish a humanized mouse model.
  • the expanded cultured A375 cells were collected and mixed with matrigel, and then inoculated subcutaneously into NOG-DKO mice in an amount of 2E6.
  • mice When the tumors grew to a volume of 100 mm 3 , mice were grouped randomly, with 8 mice in each group, and administered on the same day.
  • Group G1 was administered with an isotype control at a dose of 20 mg/kg; group G2 was administered with MHG8 (positive control) at a dose of 20 mg/kg; group G3 was administered with JYB1907hz18 at a dose of 20 mg/kg; group G4 was administered with Tencentriq at a dose of 10 mg/kg; group G5 was administered with MHG8 (at a dose of 20 mg/kg) and Tencentriq (at a dose of 10 mg/kg); group G6 was administered with JYB1907hz18 (at a dose of 20 mg/kg) and Tencentriq (at a dose of 10 mg/kg). All mice were administrated intraperitoneally twice a week for a total of 8 doses.
  • the mean tumor volume in G1 group was 1110 ⁇ 104 mm 3
  • the mean tumor volume in G2 group was 1088 ⁇ 116 mm 3
  • the mean tumor volume in G3 group was 1000 ⁇ 233 mm 3
  • the mean tumor volume in G4 group was 834 ⁇ 155 mm 3
  • the mean tumor weight in G5 group was 611 ⁇ 146 mm 3
  • the mean tumor volume in G6 group was 471 ⁇ 87 mm 3
  • the tumor inhibition rate in G5 group was 50%
  • the tumor inhibition rate in G6 group was 64%.
  • G5 and G6 groups were significantly different from G1 group (G5 vs G1, G6 vs G1), G5 vs G1, P ⁇ 0.05; G6 vs G1, P ⁇ 0.001. There was no statistical difference in other groups.
  • the combination of MHG8 with Tecentriq (anti-PD-L1 antibody) and JYB1907hz18 with Tecentriq (anti-PD-L1 antibody) were demonstrated to inhibit tumor growth, and the results were shown in Fig. 9 .
  • mice were grouped randomly, with 8 animals in each group, which were administered on the same day.
  • Group G1 was administered with an isotype control at a dose of 20 mg/kg;
  • group G2 was administered with JYB1907hz18 at a dose of 20 mg/kg;
  • group G3 was administered with Tecentriq at a dose of 10 mg/kg. All mice were administrated intraperitoneally twice a week for a total of 8 doses.
  • the mean tumor volume in G1 group was 740 ⁇ 57 mm 3
  • the mean tumor volume in G2 group was 426 ⁇ 45 mm 3
  • the mean tumor volume in G3 group was 469 ⁇ 72 mm 3
  • the tumor inhibition rate in G2 group was 52%
  • the tumor inhibition rate in G3 group was 45%.
  • Both of G2 and G3 groups were significantly different from G1 group (G2 vs G1, G3 vs G1), G2 vs G1, P ⁇ 0.001; G3 vs G1, P ⁇ 0.01.
  • mice were grouped randomly, with 8 animals in each group, which were administered on the same day.
  • Group G1 was administered with an isotype control at a dose of 20 mg/kg;
  • group G2 was administered with JYB1907hz18 at a dose of 20 mg/kg;
  • group G3 was administered with Tecentriq at a dose of 10 mg/kg. All mice were administrated intraperitoneally twice a week for a total of 8 doses.
  • the mean tumor volume in G1 group was 1273 ⁇ 212 mm 3
  • the mean tumor volume in G2 group was 797 ⁇ 113 mm 3
  • the mean tumor volume in G3 group was 842 ⁇ 115 mm 3
  • the tumor inhibition rate in G2 group was 42%
  • the tumor inhibition rate of G3 group was 38%. From the tumor growth trend graph, both JYB1907hz alone and Tecentriq (anti-PD-L1 antibody) alone tended to inhibit tumor growth on this cancer species, and the results were shown in Fig. 11 .

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